A C-terminal motif targets Hedgehog to axons, coordinating assembly of the Drosophila eye and brain

The developmental signal Hedgehog is distributed to two receptive fields by the photoreceptor neurons of the developing Drosophila retina. Delivery to the retina propagates ommatidial development across a precursor field. Transport along photoreceptor axons induces the development of postsynaptic neurons in the brain. Hedgehog is composed of N-terminal and C-terminal domains that dissociate in an autoproteolytic reaction that attaches cholesterol to the N-terminal cleavage product. Here, we show that the N-terminal domain is targeted to the retina when synthesized in the absence of the C-terminal domain. In contrast to studies that have focused on cholesterol as a determinant of subcellular localization, we find that the C-terminal domain harbors a conserved motif that overrides retinal localization, sending most of the autocleavage products into vesicles bound for growth cones or synapses. Competition between targeting signals at the opposite ends of Hedgehog apparently controls the match between eye and brain development.     

Inhibitory effect of combinations of digoxin and endogenous cardiotonic steroids on Na+/K+-ATPase activity in human kidney membrane preparation

AimsCardiac glycosides have been extensively used in the treatment of congestive heart failure for more than 200 years. Recently, cardenolides and bufadienolides were isolated from mammalian tissue and are considered as a new class of steroidal hormones. The aim of the present work was to characterize the interaction between the most clinical used cardiac glycoside digoxin and the cardiac glycosides known to exist endogenously, i.e., ouabain, marinobufagin and telocinobufagin, on human kidney Na⁺/K⁺-ATPase.Main methodsInhibition of Na⁺/K⁺-ATPase activity from crude membrane preparations of human kidney was performed using increasing concentrations of the drugs alone or mixtures of ouabain:digoxin, telocinobufagin:digoxin and marinobufagin:digoxin in a fixed ratio 1:4, 2:3 and 3:2, respectively. The colorimetric method of Fiske and Subbarow was used to measure the inorganic phosphate released.Key findingsAnalyses of inhibition curves showed that the experimental curves for all combinations were superimposed on the theoretical additive curves indicating that an additive effect occurs among distinct cardenolides and bufadienolides combinations on the human α1β1 Na⁺/K⁺-ATPase protomer.SignificanceConsidering the extensive use of digoxin in the treatment of heart failure and the recent findings that endogenous cardiac glycosides may have altered levels in many diseases, including heart failure, the demonstration of additive effect between cardiac glycosides can help in the understanding of recent clinical observations, including that lower than usual doses of cardiac glycosides are necessary for decreasing mortality in these patients.     

Analysis of oxidative stress-induced protein carbonylation using fluorescent hydrazides

Protein carbonyl detection has been commonly used to analyze the degree of damage to proteins under oxidative stress conditions. Most laboratories rely on derivatization of carbonyl groups with dinitrophenylhydrazine followed by Western blot analysis using antibodies against the dinitrophenyl moiety. This paper describes a protein carbonyl detection method based on fluorescent Bodipy, Cy3 and Cy5 hydrazides. Using this approach, Western blot and immunodetection are no longer needed, shortening the procedure and increasing accuracy. Combination of Cy3 and Cy5 hydrazides allows multiplexing analyses in a single two-dimensional gel. Derivatization with Bodipy hydrazide allows easy matching of the spots of interest and those obtained by general fluorescent protein staining methods, which facilitates excising target proteins from the gels and identifying them. This method is effective for detecting protein carbonylation in samples of proteins submitted to metal-catalyzed oxidation "in vitro" and assessing the effect of hydrogen peroxide and chronological aging on protein oxidative damage in yeast cells.     

Site-specific PEGylation of native disulfide bonds in therapeutic proteins

Native disulfide bonds in therapeutic proteins are crucial for tertiary structure and biological activity and are therefore considered unsuitable for chemical modification. We show that native disulfides in human interferon alpha-2b and in a fragment of an antibody to CD4(+) can be modified by site-specific bisalkylation of the two cysteine sulfur atoms to form a three-carbon PEGylated bridge. The yield of PEGylated protein is high, and tertiary structure and biological activity are retained.     

Protein kinase D1 inhibition interferes with mitosis progression

Protein kinase D1 (PKD1) plays a vital role in signal transduction, cell proliferation, membrane trafficking, and cancer; however, the majority of the studies up to date had centered primarily on PKD1 functions in interphase, very little is known about its role during cell division. We previously demonstrated that during mitosis PKD1 is activated and associated with centrosomes, spindles, and midbodies. However, these observations did not address whether PKD1 was associated with mitosis regulation. Accordingly, we used rapidly acting PKD-specific inhibitors to examine the contribution of PKD1 the sequence of events in mitosis. We found that although PKD1 overexpression did not affect mitosis progression, suppression of its catalytic activity by two structurally unrelated inhibitors (kb NB 142-70 and CRT 0066101) induced a significant delay in metaphase to anaphase transition time. PKD1 inhibition during mitosis also produced the appearance of abnormal spindles, defects in chromosome alignment, and segregation as well as apoptosis. Thus, these observations indicate that PKD1 activity is associated with mitosis regulation.     

Analysis of copy number variation using quantitative interspecies competitive PCR

Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility. Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts. We have developed a simple competitive PCR based method to determine DNA copy number which uses the entire genome of a single chimpanzee as a competitor thus eliminating the requirement for competitive sequences to be synthesized for each assay. This results in the requirement for only a single reference sample for all assays and dramatically increases the potential for large numbers of loci to be analysed in multiplex. In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls.     

Microbial boundstone dominated carbonate slope (Upper Carboniferous,N Spain): Microfacies,lithofacies distribution and stratal geometry

Summary The Carboniferous, particularly during the Serpukhovian and Bashkirian time, was a period of scarce shallow-water calcimicrobial-microbialite reef growth. Organic frameworks developed on high-rising platforms are, however, recorded in the Precaspian Basin subsurface, Kazakhstan, Russia, Japan and Spain and represent uncommon occurrences within the general trend of low accumulation rates and scarcity of shallow-water reefs. Sierra del Cuera (Cantabrian Mountains, N Spain) is a well-exposed high-rising carbonate platform of Late Carboniferous (Bashkirian-Moscovian) age with a microbial boundstone-dominated slope dipping from 20° up to 45°. Kilometer-scale continuous exposures allow the detailed documentation of slope geometry and lithofacies spatial distribution. This study aims to develop a depositional model of steep-margined Late Paleozoic platforms built by microbial carbonates and to contribute to the understanding of the controlling factors on lithofacies characteristics, stacking patterns, accumulation rates and evolution of the depositional architecture of systems, which differ from light-dependent coralgal platform margins. From the platform break to depths of nearly 300 m, the slope is dominated by massive cement-rich boundstone, which accumulated through the biologically induced precipitation of micrite. Boundstone facies (type A) with peloidal carbonate mud, fenestellid and fistuliporid bryozoans, sponge-like molds and primary cavities filled by radiaxial fibrous cement occurs all over the slope but dominates the deeper settings. Type B boundstone consists of globose centimeter-scale laminated accretionary structures, which commonly host botryoidal cement in growth cavities. The laminae nucleate around fenestellid bryozoans, sponges, Renalcis and Girvanella-like filaments. Type B boundstone typically occurs at depths between 20–150 m to locally more than 300 m and forms the bulk of the Bashkirian prograding slope. The uppermost slope boundstone (type C; between 0 and 20–100 m depth) includes peloidal micrite, radiaxial fibrous cement, bryozoans, sponge molds, Donezella, Renalcis, Girvanella, Ortonella, calcareous algae and calcitornellid foraminifers. From depths of 80–200 m to 450 m, 1–30 m thick lenses of crinoidal packstone, spiculitic wackestone, and bryozoan biocementstone with red-stained micrite matrix are episodically intercalated with boundstone and breccias. These layers increase in number from the uppermost Bashkirian to the Moscovian in parallel with the change from a rapidly prograding to an aggrading architecture. The red-stained strata share comparable features with Lower Carboniferous deeper-water mud-mound facies and were deposited during relative rises of sea level and pauses in boundstone production. Rapid relative sea-level rises might have been associated with changes in oceanographic conditions not favourable for thecalcimicrobial boundstone growth, such as upwelling of colder, nutrient-rich waters lifting the thermocline to depths of 80–200 m. Downslope of 150–300 m, boundstones interfinger with layers of matrix-free breccias, lenses of matrix-rich breccias, platform- and slope-derived grainstone and crinoidal packstone. Clast-supported breccias bound by radiaxial cement are produced by rock falls and avalanches coeval to boundstone growth. Matrix-rich breccias are debris flow deposits triggered by the accumulation of red-stained layers. Debris flows develop following the relative sea-level rises, which favour the deposition of micrite-rich lithofacies on the slope rather than being related to relative sea-level falls and subaerial exposures. The steep slope angles are the result of in situ growth and rapid stabilization by marine cement in the uppermost part, passing into a detrital talus, which rests at the angle of repose of noncohesive material. In the Moscovian, the aggradational architecture and steeper clinoforms are the result of increased accommodation space due to tectonic subsidence and due to a reduction of slope accumulation rates (from 240±45−605±35 m/My to 130±5 m/My). The increasing number of red-stained layers and the decrease of boundstone productivity are attributed to environmental changes in the adjacent basin, in particular during relative rises of sea level and to possible cooling due to icehouse conditions. The geometry of the depositional system appears to be controlled by boundstone growth rates. During the Bashkirian, the boundstone growth potential is at least 10 times greater than average values for ancient carbonate systems. The slope progradation rates (nearly 400–1000 m/My) are similar to the highest values deduced for the Holocene Bahamian prograding platform margin. The fundamental differences with modern systems are that progradation of the microbial-boundstone dominated steep slope is primarily controlled by boundstone growth rates rather than by highstand shedding from the platform top and that boundstone growth is largely independent from light and controlled by the physicochemical characteristics of seawater.     

Sex-sorting of boar spermatozoa does not influence the localization of glucose transporters

Sex-sorting damages spermatozoa function, shortening their lifespan and fertility. This study used an immunofluorescence technique to investigate the effect of sex-sorting on the localization of glucose transporters (GLUTs) in boar spermatozoa. GLUTs are trans-membrane proteins responsible for glucose transport within cells. Distribution of GLUTs on sperm cells was similar in unsorted and sex-sorted semen, suggesting that the flow cytometric sex-sorting process did not affect the sperm energy apparatus.     

Targeting of a T cell agonist peptide to lysosomes by DNA vaccination induces tolerance in the nonobese diabetic mouse

CD4 T cells are crucial effectors in the pathology of type 1 diabetes (T1D). Successful therapeutic interventions for prevention and cure of T1D in humans are still elusive. Recent research efforts have focused on the manipulation of T cells by treatment with DNA. In this paper, we studied the effects of a DNA treatment strategy designed to target antigenic peptides to the lysosomal compartment on a monospecific T cell population termed 2.5mi(+) T cells that shares reactivity with the diabetogenic T cell clone BDC-2.5 in the NOD mouse. MHC class II tetramer analysis showed that repeated administrations were necessary to expand 2.5mi(+) T cells in vivo. This expansion was independent of Ag presentation by B cells. A single peptide epitope was sufficient to induce protection against T1D, which was not due to Ag-specific T cell anergy. Typical Th2 cytokines such as IL-10 or IL-4 were undetectable in 2.5mi(+) T cells, arguing against a mechanism of immune deviation. Instead, the expanded 2.5mi(+) T cell population produced IFN-γ similar to 2.5mi(+) T cells from naive mice. Protection against T1D by DNA treatment was completely lost in NOD.CD28(-/-) mice which are largely deficient of natural regulatory T cells (Treg). Although Ag-specific Foxp3(+) Treg did not expand in response to DNA treatment, diabetes onset was delayed in Treg-reconstituted and DNA-treated NOD.SCID mice. These observations provide evidence for a Treg-mediated protective mechanism that is independent of the expansion or de novo generation of Ag-specific Treg.